The molecular chaperone that is known as a heat shock protein functions to prevent incorrect structure forming that is resulted from the interaction between subdomains of a polypeptide or between a polypeptide and other molecules. Although molecular chaperone mediates the perfect assembling of a polypeptide, it has nothing to do with the assembled protein because it is no more a subunit of a protein once the assembling procedure is finished.
The molecular chaperone helps a polypeptide assemble itself by obstructing alternative assembly pathways forming a nonfunctional structure and mediates folding of a newly synthesized polypeptide chain. The molecular chaperone is separated from a protein once the protein matrix has not a misfolding conformation any more. These molecular chaperones are characterized by the ability to bind a partly folded intermediary metabolite temporarily during the folding and assembling procedure of a protein. Precisely, they do not bind to a completely folded and assembled conformation and function to make an effective folding and assembling procedure by preventing premature folding and gathering intermediary metabolites.
As explained hereinbefore, the molecular chaperone has been confirmed to help a protein have a normal stable confirmation by preventing misfolding of proteins largely. Thus, the molecular chaperone is confirmed to be an important factor to promote the productivity of proteins.
Recently, lots of medical supplies using recombinant proteins are being produced. Particularly, such medical supplies are produced by transfecting gene of interest into host cells. For the stable market of medical supplies, it is important to mass-produce a recombinant protein, so called a target protein, which triggers vigorous studies on the way to promote the productivity of foreign proteins from many host cell lines. As an example, studies are undergoing to promote the productivity of foreign proteins using CHO cell line. The final concentration of target proteins produced from CHO cell line is affected by the density of live cells producing target proteins and specific production rate as seen in the below <Mathematical Formula 1>.Final protein concentration=Cell density×Specific production rate  <Mathematical Formula 1>
It has been a major study object to increase cell density by taking advantage of batch culture for culturing cells producing target proteins since specific production rate has been regarded to be a native factor to a cell and not to be changed. However, in order to increase specific production rate, methods to increase production speed of cells using high osmotic pressure media or gene amplification system like dihydrofolate reductase(dhfr)/methotrexate(MTX) and glutamine synthatase(GS)/methionine sulfoximine(MSX) have been tried. But those methods are to increase the concentration of final product by just making the best of either cell density determining the concentration of target protein or specific production rate. Besides, when gene amplification system is used, the copy number of recombinant gene increases only to a limited level and thus, the productivity of recombinant protein does not increase further over the level, resulting in just saturation. When high osmotic pressure medium is used, specific production rate goes up, though, cell growth and survival rate decreases. The reason why the protein productivity is in saturation even though the copy number of gene increases continuously seems to be because of speed determining step of endoplasmic reticulum (Fred J. Stevens and Yair Argon, Cell & Developmental Biology, 1999, 10, 443-454). So an attempt to introduce molecular chaperone, a factor determining protein secretion speed in endoplasmic reticulum, to cells producing target proteins has been made in order to increase over-expression of chaperone therein. But over-expression of chaperone rather brought negative effects. For example, over-expression of PDI protein in CHO cells held TNFR:Fc in endoplasmic reticulum, resulting in the decrease of secretion thereof (Raymond Davis, et al., Biotechnology Progress, 2000, 16, 736-743), and over-expression of GRP78 in CHO cells inhibited the secretion of mutant forms of von Willebrand factor and factor VIII (Dorner, A. J. et al., EMBO J., 1992, 11, 1563-1571).
Thus, another attempt to use a gene expression regulating system has been made again. Among many gene expression-regulating systems, Tet-Off system has been widely used because the system has fewer side effects and can regulate gene expression selectively. Tet-Off system is based on a regulating element of tetracycline resistant operon of Escherichia coli. This operon comprises transactivator (tTA) regulated by tetracycline and a promoter depending on tTA. The promoter does not work at all when tetracycline (ex: doxycycline or mynocycline) is there and is only activated in the absence of tetracycline (FIG. 1). Tet-Off system draws an attention since gene expression in mammalian cells can be regulated, the most exact on/off regulation is possible and the regulation with inexpensive tetracycline or doxycycline is also possible with the system, etc.
Thus, the present inventors completed this invention by confirming the fact that the production of target protein is regulated or the mass-production thereof is possible by regulating the expression of chaperone in cells producing target protein by introducing Tet-Off system that regulates the expression of chaperone known to help the protein secretion in endoplasmic reticulum.